The human adrenal cortex can be divided into distinct zones that have both morphologic and biochemical differences. The production of aldosterone in the zona glomerulosa and cortisol in the zona fasciculate can be traced to the zone-specific expression of the enzymes involved in steroid biosynthesis. This is particularly true for aldosterone synthase (CYP11B2) and 112-hydroxylase (CYP11B1). Our preliminary data suggest that three nuclear hormone receptors play pivotal roles in determining a glomerulosa versus fasciculata phenotype. Herein, we test the overall hypothesis that zone-specific expression of CYP11B2 and CYP11B1 results from the opposing activities of nuclear hormone receptors that differ between the glomerulosa and fasciculata. This hypothesis will be tested by the studies proposed in four Specific Aims: The experiments proposed in Specific Aim One will extend the applicant's ongoing project that has defined the regulatory elements in the 5'-flanking region of CYP11B2 gene. The proposed studies will complete the definition of the trans-acting factors responsible for glomerulosa specific expression of CYP11B2. Focus will be placed on the role of nuclear receptor, NURR1 (NR4A2), which stimulates CYP11B2 promoter activity, is up-regulated by angiotensin II and is expressed primarily in the zona glomerulosa. Specific Aim Two will determine the mechanisms responsible for the lack of CYP11B2 seen in the glomerulosa. Preliminary studies indicate that the nuclear receptor, steroidogenic factor 1 (SF-1; NR5A1) is a potent repressor of CYP11B2 but potent activator of CYP11B1. These data support a role for SF-1 activity in the transition of glomerulosa to fasciculata. Specific Aim 3 will determine the role of nuclear hormone receptor post-translational modification in the development of a glomerulosa cell phenotype. Preliminary data indicate that the glomerulosa has elevated sumoylation activities and sumoylation of SF-1 regulates its activity. Specific Aim 4 will define the cis-regulatory elements and trans-acting factors responsible for silencing CYP11B2 expression in the fasciculata. We have isolated a 100 bp insert that is present in the CYP11B2 but not CYP11B1 gene. Preliminary data support the possibility that this insert includes a silencer sequence that acts to block fasciculata cell expression of CYP11B2. SIGNIFICANCE: Primary aldosteronsism (PA) is estimated to occur in 8 % of the hypertensive population. Most PA cases are caused by nodular hyperplasia or aldosterone-producing adenoma. A disruption in the normal regulation of CYP11B2 expression appears to be an important part of this disease process. The proposed studies will provide a detailed understanding of the molecular mechanisms regulating CYP11B2 expression within the normal adrenal and should provide insight into diseases of aldosterone excess including hyperaldosteronism. PUBLIC HEALTH RELEVANCE: Narrative Information obtained from these studies will determine the molecular mechanisms causing zone- specific expression of CYP11B2 and CYP11B1 thereby advancing our understanding of adrenal zonation. In addition, the proposed studies will help to better define the causes of diseases of adrenal steroid excess including hyperaldosteronism.